A. Nerve Growth Factor (NGF)
The nerve growth factor (NGF) was first discovered in mouse sarcoma tumors (Levi-Montalcini, R. et al., J. Exp. Zool. 116:321, 1951) and was then purified and brought to homogeneity from male mouse salivary submaxillary glands (Varon, S. et al., Biochemistry 6:2202, 1967) and from snake venom (Angeletti, R. H., Proc. Natl. Acad. Sci. USA 65:668, 1970). Many other relatively rich NGF sources have been indicated, including guinea pig prostate (Harper, G. P. et al., Nature 279:160, 1979) and human placenta (Goldstein, L. D. et al., Neurochem. Res. 3:175, 1978, Walker, P. et al., Life Science 26:195, 1980, Fidia Patent 47745A88). Small amounts of NGF have been found in other tissues such as, for example, the mammal central nervous system (Whittemore, Scott, R. et al., Brain Research 12, 439-464, 1987. The physiological relation between these potential sources of NGF and the apparent action sites is not very clear but generally it is supposed that NGF is secreted by various peripheral tissues that require innervation from those cells that respond to the NGF.
The NGF obtained from the mouse submaxilliary glands is the one mostly used for studies of the activity of NGF in vitro and in vivo. The range of biological activity in vitro of NGF was determined both on primary nerve cells and on cloned cell lines. Of the primary nerve cells that responded to the NGF in vitro are fetal sensory neurons (fetal day 8-12) from the spinal ganglion roots, autonomic noradrenergic fetal neurons from the sympathetic ganglion, cholinergic fetal neurons from the septum and chromaffin suprarenal cells during development. While the sensory and sympathetic neurons depend on NGF to survive and develop, the cholinergic neurons do not seem to need NGF for survival but only for their differentiation, i.e., for expression of the phenotypic characteristics linked to the neurotransmitter. The addition of NGF to the chromaffin suprarenal cells (derived from the neural crest) during the first phase of their development causes the expression of nerve phenotypes. Of the cells lines that respond to the NGF in vitro, as described in the literature, included are the chromaffin suprarenal cells derived from tumors of the neural crest, called pheochromocytoma cells (PC12) and human neuroblastoma cells. After treatment with .beta.-NGF, these cells change their behavior, going from a strongly proliferative phase to a postmitotic condition.
The nerve growth factor obtained from mouse submaxillary gland is the one which is most characterized, also with a chemical and immunochemical profile. The NGF from murine glands acts like a protein complex of the 7S type (molecular weight about 140,000 daltons) made up of three subunits (.alpha., .beta., .gamma.) that coordinate a Zn.sup.+ atom.
The most interesting part of the 7S molecule, relative to biological activity, is constituted by two polypeptide chains, each having a molecular weight of 13,250 and formed by 118 amino acids. Each chain or monomer has three sulfide bridges, that form covalent bonds between two cysteine residues, which confer a strong stability to the tridimensional structure of the protein. The two monomers of NGF joined to one another by weak bonds form a dimer with molecular weight of 26,500. It has been shown that the biological activity is associated with the dimer called 2.5S or conventionally .beta.-subunit. It is not known if this is present also in the monomer.
The techniques of genetic engineering have made it possible to identify the gene that codes for the .beta.-subunit of NGF (.beta.-NGF) (Scott, J. et al., Nature 302:538, 1983; Ullrich, A. et al., Nature 303:821, 1983; EP Patent Publn. No. 0 121 338). The human gene that codes the molecule is located in the short arm of chromosome I and codes for the synthesis of a molecule much larger than that of molecular weight of 26,500 that constitutes the biologically active molecule. Therefore, the gene initially instructs the synthesis of a NGF precursor or pro-NGF of greater dimensions. It has further been demonstrated that the gene coding for the .beta.-subunit of NGF is highly conserved in different species, from birds to man (Meier, R. et al., EMBO J. 5:1489, 1986).
The elucidation of the nucleotide sequences of murine, human, bovine and chick .beta.-NGF has made possible a comparison between the conserved sites and those not conserved of these molecules and their relationship to biological activity and antigenicity. The overall conservation of .beta.-NGF during the evolution is surprisingly high. Of 118 amino acids of the mature form of NGF purified from male mouse salivary submaxillary glands, only 16 amino acids are different in bovine .beta.-NGF, 19 in chick .beta.-NGF and 11 in human .beta.-NGF, while there are only 6 amino acids of difference between bovine and human .beta.-NGF. All the cysteine residues are rigorously conserved in all species. The reduction of the three S-S bridges of .beta.-NGF causes the complete loss of its biological activity. The apparent discrepancy between the high level of overall conservation of amino acid sequences and the low cross-reactivity of the immunochemical type is due to the fact that the changes of the amino acids between species are located in specific "clusters." With hydropathic tracings it is possible to demonstrate that these changes occur almost completely in hydrophilic sites considered as potential antigenic determinants. Only one hydrophilic region was seen to be strictly conserved in the NGF molecules for all species studied so far.